The enzymatic hydrolysis of organic nitrites to corresponding carboxylic acids and amides provides an important alternative synthetic method to a broad spectrum of useful compounds. Conventional chemical hydrolysis of nitrites to the corresponding carboxylic acids and amides is typically carried out using a strong acid or base catalyst at high reaction temperatures making it incompatible with compounds which contain sensitive functional groups. Furthermore, the poor selectivity of chemical hydrolysis may result in unwanted by-products along with large quantities of inorganic salts. In contrast, enzymatic nitrile hydrolysis occurs under mild conditions (neutral pH, 30° C.) offering the potential for high chemo-, regio-, and stereoselectivity. As an added advantage, the formation of by-product inorganic salts is avoided.
The best-known industrial applications of nitrile-converting enzymes are the production of acrylamide (T. Nagasawa et al., Tibtech., 1992, vol. 10, 402-408) and nicotinamide (T. Nagasawa et al., Appl. Environ. Microbiol., 1998, vol 54, 1766-1769), using a nitrile hydratase from Rhodococcus rhodochrous J1. Several recent reviews (L. Martinková et al., Current Organic Chemistry, 2003, vol. 7, 1279-1295 and D. Cowan et al., Extremophiles, 1998, vol. 2, 207-216) describe the biochemistry and potential industrial applications of nitrile converting enzymes.
Enzymatic nitrile hydrolyses are catalyzed by nitrilases, which convert nitrites to the corresponding carboxylic acids, and nitrile hydratases, which convert nitrites to the corresponding amides. Amidases, which hydrolyze amides to the corresponding carboxylic acids, can be used in combination with nitrile hydratases to convert nitrites to carboxylic acids.
The use of a nitrilase enzyme to prepare a carboxylic acid from the corresponding nitrile is disclosed in WO 02/072856. Incorporation of the enzyme into a polymer matrix with cross-linking provided a catalyst with improved physical and biochemical integrity.
The regioselective preparation of ω-nitrilecarboxylic acids from aliphatic ∝, ω-dinitriles with a biocatalyst was disclosed in U.S. Pat. No. 5,814,508. For example, a catalyst having nitrilase activity was used to convert 2-methylglutaronitrile into 4-cyanopentanoic acid.
K. Yamamoto, et al. J. Ferment Bioengineering, 1992, vol. 73, 125-129 describes the use of microbial cells having both nitrile hydratase and amidase activity to convert trans 1,4-dicyanocyclohexane to trans-4-cyanocyclohexanecarboxylic acid.
Regioselective biocatalytic conversions of dinitriles to cyano substituted carboxylic acids, have been reported for a series of aliphatic α, ω-dinitrile compounds using microbial cells having an aliphatic nitrilase activity or a combination of nitrile hydratase and amidase activities (J. E. Gavagan et al. J. Org. Chem., 1998, vol. 63, 4792-4801).
Stereoselective enzymatic conversions of nitriles have been described for the preparation of chiral carboxylic acids and amides enriched in one enantiomer (M Wieser et al., Chapter in Stereoselective Biocatalysis, Marcel Dekker Inc.: New York, 2000, 461-486). A stereoselective nitrilase enzyme from Alcaligenes faecalis ATCC 8750 is used to prepare (R)-mandelic acid from racemic mandelonitrile (K. Yamamoto et al., Appl. Environ. Microbiol., 1991, vol. 57, 3028-3032). A nitrilase from Rhodococcus rhodochrous NCIMB 11216 preferentially hydrolyzes (+)-2-methylhexanitrile in a racemic mixture of 2-methylhexanitrile leaving (−)-2-methylhexanitrile unreacted (M. Gradley et al., Biotechnology Lett., 1994, vol. 16, 41-46). U.S. Pat. No. 5,593,871 disclosed a process for preparing 2-alkanoic acid amides enriched in one enantiomer, from nitriles using microorganisms containing stereoselective nitrile hydratases. Enantiopure α-amino acids and amides were prepared from racemic α-aryl and α-alkyl-substituted glycine nitriles using Rhodococcus sp. AJ270 containing a stereoselective nitrile hydratase and a stereoselective amidase (M.-C. Wang et al., J. Org. Chem., 2002, vol. 67, 6542). The foregoing references are hereby incorporated herein in their entirety.
The therapeutic value of racemic pregabalin, particularly its efficacy as an anticonvulsant, has been found to be attributable primarily to the (S)-enantiomer. Toward the goal of providing cost-effective pregabalin drug therapy, a number of synthetic routes to the (S)-enantiomer enriched compound have been investigated. For example, asymmetric hydrogenation of the appropriate cyano substituted olefin followed by reduction of the cyano group to the corresponding amine provides pregabalin substantially enriched in the (S) enantiomer (United States Patent Application Publication No. 2003/0212290).
The synthesis of pregabalin, its derivatives and analogs by purely chemical methods is disclosed in U.S. Pat. Nos. 6,642,398; 6,635,673; and 6,046,353.